The agouti locus (a) in chromosome 2 regulates the differential production of black and yellow pigment granules that give rise the agouti coat color of the mouse. Agouti coloration, which is the true wild-type coat color of mice, is unusual in that it arises not from a homogenous pigmentation of the pelage, but rather from a banded coloration pattern in which each hair is black with a subapical band of yellow. One of the most interesting aspects of the agouti locus is that it functions within the microenvironment of the hair follicle (Silver, W. K. and Russell, 1955, J. Exp. Zool., 130: 199-220; Silvers, W. K. 1958, J. Exo. Zool. 137: 181-188; Silvers, W. K. 1958, J. Exo. Zool. 137: 189-198; Silvers, W. K. 1961 Science 134: 368-373; Silvers, W. K. 1979 in: The Coat. Colors of Mice: A Model for Mammalian Gene Action and Interaction, New York, N.Y., Springer-Verlag, pp. 6-44), unlike Hay other coat color genes, which act in a cell-autonomous manner within the melanocytes. Therefore, agouti must be regulating coat pigmentation by some direct or indirect form of intercellular signaling within the follicular environment.
Like many other genes that play a role in the regulation of coat pigmentation in the mouse, the agouti locus contributes to essential developmental processes unrelated to pigmentation (Geissler, E. N. et al., 1988, Cell 55: 185-192; Witte, O. N. 1990 Cell 63: 5-6; Epstein, D. J. et al. 1991 Cell 67: 767-774; Mercer, J. A. et al. 1991 Nature 349: 709-713). For example, some of the individual alleles at the agouti locus are associated with embryonic lethality, obesity, diabetes, and the development of tumors in a wide variety of tissues. In fact, the lethal yellow (A.sup.y) mutation at agouti was the first embryonic lethal mutation to be characterized in the mouse (Cuenot, L. 1905 Arch. Zool. Exp. Gen. 3: 123-132). Embryos homozygous for Ay die very early in development, around the time of implantation, possibly owing to a defect in trophectoderm differentiation (Eaton, G. J. and Green, M. M. 1963 Genetica 34: 155-161; Calarco, P. G. and Pederson, R. A. 1976; Papaioannou, V. E. and Gardner, R. L. 1979 J. Embryol. Exp. Morphol. 52: 153-163).
Genetic analyses of numerous a locus mutants have been ongoing for nearly a century, and have led to the identification of at least 18 dominant and recessive alleles and seudoalleles of agouti (Silvers, W. K. 1979 ibid; Green, M. C. 1989. In: Genetic Variants and Strains of the Laboratory Mouse, M. F. Lyon and A. G. Searle, eds., Oxford, Oxford University Press, pp. 17-20). Different combinations of alleles account for an array of different phenotypes, ranging from subtle differences in coat color as compared with the wild type, to drastic changes in the distribution of pigmentation in different regions of the animal, particularly across the dorso-ventral surface. An intricate dominance hierarchy exists in which alleles associated with phaeomelanin (yellow) production are generally dominant over alleles associated with eumelanin (Black or brown, depending on alleles at other loci) production. This relationship is exemplified by several alleles that date back to the mouse fancy: lethal yellow (A.sup.y), which confers an all-yellow phenotype in the heterozygous condition, black-and-tan(a.sup.t), which gives rise to an all-black dorsum and an all-yellow ventrum (Dunn, L. C. 1928 Proc. Natl. Acad. Sci. USA 14: 816-819), nonagouti (a), which gives rise to a predominantly black phenotype, except for small amounts of phaeomelanin around the pinnae, nipples, and perineum, and extreme nonagouti (a.sup.c), which confers a completely black phenotype (Hollander, W. F. and Gowen, J. W. 1956 J. Hered. 47: 221-224).
The large number of alleles and the wide range of phenotypes associated with the agouti locus have been used as evidence by some investigators to propose that the agouti locus is comprised of multiple "mini-loci" and not a single gene. According to this hypothesis, each gene of the mini-locus plays a role in regulating pigmentation in different parts of the body, particularly over the dorsal and ventral surfaces, and around the pinae, nipples, and perineum. Support for this assertion stems from the finding that changes from yellow to black pigmentation proceed from the dorsal to the ventral regions as one progresses from the most dominant to the most recessive mutation of the agouti allelic series. For example, phaeomelanin progressively disappears from the mid-dorsum with A.sup.i /a (A.sup.i, intermediate yellow), from the lateral dorsum with a.sup.t /a.sup.t, from the ventral surface with a/a, and from the pinnae, nipples, and perineum with a.sup.c. With the mini-locus hypothesis, different genes should be affected by mutations associated with the individual alleles in the hierarchy. The present invention demonstrates that the structure and expression of the same gene is affected by mutations at the top (A.sup.y), middle (a.sup.t and a), and bottom (a.sup.c) of the allelic series. These results disprove the mini-locus hypothesis.
Although the agouti alleles have been extensively characterized with classical genetic techniques, the structure of the gene(s) responsible for a locus function had not been determined until the present invention. Attempts by others to isolate the gene using positional cloning techniques, failed to isolate the gene (Barsh and Epstein 1989a. Genomics 121: 811-818; Siracusa et al. 1987 a Genetics 117: 93-100;,Siracusa et al. 1989 Genetics 122: 669-679; Siracusa 1991. Ann. N.Y. Acad. Sci 642: 419-430.).
A radiation-induced inversion mutation, called Is(17; In2)Id, aJGso (abbreviated Is1Gso), which contains DNA breakpoints in the limb deformity (1d) and agouti loci, two regions that are normally separated by 22 cM on chromosome 2 (Woychik, R. P. et al. 1990a Proc. Natl. Acad. Sci., USA 87: 2588-2592; Bultman, S. J. et al. 1991 Proc. Natl. Acad. Sci., USA 88: 8062-8066) was previously described. Utilizing a DNA probe from the Id.sup.Hd insertional mutant (Woychik, R. P. et al. 1985 Nature 318: 36-40), 22 cM were jumped with the inversion which allowed a region of DNA that maps to the agouti locus to be identified (Woychik et al. 1990a, ibid). Moreover, this region also hybridizes to sequences that are rearranged in several agent-induced a locus mutations (Bultman, S. J. et al. 1991, ibid). Here we present the molecular characterization of a gene from this region of DNA that is structurally altered in several a locus mutations and is expressed in a manner appropriate for agouti function. The present invention demonstrates that there is one gene associated with the a locus.